EUBACTERIAL tmRNA SEQUENCES AND USES THEREOF

ABSTRACT

The present invention is directed to eubacterial tmDNA sequences and the corresponding tmRNA sequences. The present invention is further directed to alignments of eubacterial tmDNA sequences and the use of the sequences and sequence alignments for the development of antibacterial drugs. The present invention is also directed to the use of the sequences for the development of diagnostic assays.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is division of U.S. patent application Ser. No.11/329,230 filed on 11 Jan. 2006, which in turn in a division of U.S.patent application Ser. No. 09/958,206 filed on 20 Feb. 2002, now U.S.Pat. No. 7,115,366, which in turn is a national stage filing under 35U.S.C. §371 of International patent application No. PCT/US00/08988 filedon 6 Apr. 2000, which in turn is related to and claims priority under 35U.S.C. § 119(e) to U.S. provisional patent application Ser. No.60/128,058 filed on 7 Apr. 1999. Each of these applications isincorporated herein by reference.

This application was made with Government support under Grant No. GM48152, funded by the National Institutes of Health, Bethesda, Md. TheGovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

The present invention is directed to eubacterial tmDNA sequences and thecorresponding tmRNA sequences. The present invention is further directedto alignments of eubacterial tmDNA sequences and use of the sequencesand sequence alignments for the development of antibacterial drugs. Thepresent invention is also directed to the use of the sequences for thedevelopment of diagnostic assays.

The publications and other materials used herein to illuminate thebackground of the invention or provide additional details respecting thepractice are incorporated by reference, and for convenience arerespectively grouped in the appended List of References.

Eubacterial tmRNAs (10Sa RNAs) are unique since they function, at leastin E. coli, both as tRNA and as mRNA (for a review, see Muto et al.,1998). These ≈360±10% nucleotide RNAs are charged with alanine at their3′-ends (Komine et al., 1994; Ushida et al., 1994) and also have a shortreading frame coding for 9 to 27 amino acids depending on the bacterialspecies. E. coli tmRNA mediates recycling of ribosomes stalled at theend of terminatorless mRNAs, via a trans-translation process (Tu et al.,1995; Keiler et al., 1996; Himeno et al., 1997). In E. coli, this aminoacid tag is co-translationally added to polypeptides synthesized frommRNAs lacking a termination codon, and the added 11 amino acidC-terminal tag makes the protein a target for specific proteolysis(Keiler et al., 1996).

Structural analyses based on phylogenetic (Felden, et al., 1996;Williams and Bartel, 1996) and probing (Felden et al., 1997; Hickersonet al., 1998) data have led to a compact secondary structure modelencompassing 6 helices and 4 pseudoknots. tmRNAs have some structuralsimilarities with canonical tRNAs, especially with tRNA acceptorbranches. E. coli tmRNA contains two modified nucleosides,5-methyluridine and pseudouridine, located in the tRNA-like domain ofthe molecule, in a seven-nucleotide loop mimicking the conservedsequence of T loops in canonical tRNAs (Felden et al., 1998).

Fifty-three tmRNA sequences are now known from both experimental dataand Blast searches on sequenced genomes (summarized in Williams, 1999;Wower and Zwieb, 1999). These sequences cover only 10 phyla, less thanone third of the known bacterial taxa. It is desired to determineadditional tmRNA sequences and to use the tmRNA sequences for drugdevelopment.

SUMMARY OF THE INVENTION

The present invention relates to eubacterial tmDNA sequences and thecorresponding tmRNA sequences. The present invention further relates toalignments of eubacterial tmDNA sequences and use of the sequences andsequence alignments for the development of antibacterial drugs.

In one aspect of the present invention, an extensive phylogeneticanalysis was performed. Fifty-eight new tmDNA sequences includingmembers from nine additional phyla were determined. Remarkably, tmDNAsequences could be amplified from all species tested apart from those inthe alpha-Proteobacteria. This aspect of the invention allowed a moresystematical study of the structure and overall distribution of tmRNAwithin eubacteria

In a second aspect of the invention, alignments are made with the newlyisolated tmDNA sequences and previously disclosed tmRNA sequences.

In a third aspect of the invention, the alignments of the tmRNAsequences allow the identification of targets for development ofantibacterial drugs.

In a fourth aspect of the invention, the novel tmDNA or tmRNA sequencesof the present invention are used to develop diagnostic assays, such asamplification-based assays, for the bacterial species disclosed herein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B show the effect of the annealing temperature (FIG. 1A) andmagnesium concentration (FIG. 1B) on amplifying eubacterial tmRNA genesfrom genomic DNAs using PCR. A: Varying the annealing temperature from50° to 70° C. during the PCR amplification of Thermus aquaticus (1). B;Varying the magnesium concentration to amplify tmDNA genes from Thermusaquaticus (1), negative effect of increasing the magnesiumconcentration), Acholeplasma laidlawii (2), positive effect ofincreasing the magnesium concentration, the upper band is the tmDNAgene) and from Mycoplasma salivarium (3), no discernible effect ofmagnesium ions in that concentration range). The arrows point toward the4 novel tmDNA genes that have been sequenced.

FIG. 2 shows the distribution of tmDNA sequences within eubacterialgenomes. The circled phyla or subgroups contain tmDNA sequences andthose shaded are new members of this category. The numbers shown closeto each phylum are the 51 tmDNA sequences that have are disclosed hereinand the numbers in parenthesis are the 53 tmDNA sequences that werepreviously known (summarized in Williams, 1999; Wower and Zwieb, 1999).The environmental samples are indicated with a dashed line as theirconnection to the tree is unknown. The 5 alpha-Proteobacteria in whichtmDNA sequences were not detected by PCR analysis are labeled “PCR” andthe 3 analyzed by Blast search of the complete, or nearly complete,sequenced genomes are labeled “database”.

FIGS. 3A, 3B and 3C show the sequence alignment, structural domains andstructural features for the tmRNA of several species of Firmicutes. ThetmRNA sequences are set forth in SEQ ID NOs:67-87.

FIGS. 4A and 4B show the sequence alignment, structural domains andstructural features for the tmRNA of several species of Thermophiles.The tmRNA sequences are set forth in SEQ ID NOs:88-99.

FIGS. 5A and 5B show the sequence alignment, structural domains andstructural features for the tmRNA of several species of Cyanobacteries(5A) and chloroplasts (5B). The tmRNA sequences of the Cyanobacteriesare set forth in SEQ ID NOs:100-103, and the tmRNA sequences of thechloroplasts are set forth in SEQ ID NOs:104-108.

FIGS. 6A and 6B show the sequence alignment, structural domains andstructural features for the tmRNA of several species of Mycoplasmes. ThetmRNA sequences are set forth in SEQ ID NOs:109-117.

FIGS. 7A-1, 7A-2, 7B, 7C and 7D show the sequence alignment, structuraldomains and structural features for the tmRNA of several species ofMesophiles (7A-1, 7A-2, 7C, 7D) and environmental sludge (7B). The tmRNAsequences of the Mesophiles are set forth in SEQ ID NOs:118-123 and125-128, and the tmRNA sequence of the environmental sludge is set forthin SEQ ID NO:124.

FIGS. 8A and 8B show the sequence alignment, structural domains andstructural features for the tmRNA of several species of Actinobacteries(8A) and Spirochaetes (8B). The tmRNA sequences of the Actinobacteriesare set forth in SEQ ID NOs:132-136, and the tmRNA sequences of theSpirochaetes are set forth in SEQ ID NOs:137-142.

FIGS. 9A and 9B show the sequence alignment, structural domains andstructural features for the tmRNA of several species of Pourpres beta.The tmRNA sequences are set forth in SEQ ID NOs:143-154.

FIGS. 10A, 10B and 10C show the sequence alignment, structural domainsand structural features for the tmRNA of several species of Pourpresgamma. The tmRNA sequences are set forth in SEQ ID NOs:155-169.

FIGS. 11A and 11B show the sequence alignment, structural domains andstructural features for the tmRNA of several species of Pourpres delta(11A) and Pourpres epsilon (11B). The tmRNA sequences of the Pourpresdelta are set forth in SEQ ID NOs:170-172, and the tmRNA sequences ofthe Pourpres epsilon are set forth in SEQ ID NOs:173-175.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to eubacterial tmDNA sequences and thecorresponding tmRNA sequences. The present invention is further directedto alignments of eubacterial tmDNA sequences and use of the sequencesand sequence alignments for the development of antibacterial drugs.

The novel eubacterial tmDNA sequences determined in accordance with thepresent invention are set forth in Tables 1-58, below. The alignment oftmRNA sequences is shown in FIGS. 3A-11B, which also show the structuraldomains and structural features of the tmRNA. The present invention alsoincludes the tmRNA sequences set forth in these figures to the extentthey differ from the sequences set forth in Tables 1-58.

The sequences, especially as identified by the sequence alignment,represent targets for the development of drugs which may be broadlyapplicable to many kinds of bacteria, or may be broadly applicable onlyto a particular genera of phylum of bacteria, or may be specificallyapplicable to a single species of bacteria. Thus, the present inventionis further directed to the development of drugs for the therapeutictreatment of bacteria, generically or specifically. Suitable drugs aredeveloped on the basis of the tmRNA sequences as described herein.

For all the novel tmRNA sequences, as well as with the ones that arealready known, there are systematically several structural domains thatare always found. These domains can be used as targets for thedevelopment of drugs which may be genera specific or which may beeubacteria specific. These domains are either RNA helices which can besometimes interrupted by bulges or pseudoknots. The RNA helices whichare always present are H1, H2, H5 and H6. Helices H1 and H6 are found inall canonical transfer RNAs. Thus, H1 and H6 are not good targets fordrug development because drugs that would target them will alsointerfere with the biology of the individual that has a given disease.Consequently, very good candidates for development of drugs fortargeting as many bacteria as possible are helices H2 and H5. Moreover,helices H2 and H5 are critical for the folding of all these tmRNA sinceboth of them connect the two ends of the molecule together. Disruptionof either H2 or H5 with a specific drug would lead to inactive tmRNAmolecules in vivo. Similarly, pseudoknots PK1, PK2 and PK3 are alwaysfound in the bacterial tmRNAs. Since these pseudoknots are not found inall canonical transfer RNAs, they can also be targeted with specificdrugs. Disruption of either PK1 or PK2 or PK3 with a specific drug wouldlead to inactive tmRNA molecules in vivo.

In addition to developing drugs which broadly target many bacteria,drugs are developed which are more genera specific. For trying to targetspecifically a given bacteria or a complete phylum, the coding sequence(shown in all the alignments) is a very good candidate. Indeed, thisregion of the RNA is very accessible for DNA antisense binding (such asshown for Escherichia coli; Matveeva et al., 1997), and thus, is alsoavailable for interaction with other drugs. Moreover, the codingsequence is a critical functional domain of the molecule in itsquality-control mechanism in cells.

Interestingly, some structural domains are present only in a givenbacterial phyla and could be targeted for discovering a drug that willbe specific of a phylum, but not of the others. For example:

(1) in the cyanobacteria, the fourth pseudoknot PK4 is made of twosmaller pseudoknots called PK4a and PK4b;

(2) in the mycoplasma, helix H2 is made of only 4 base-pairs instead of5 in the other species;

(3) for two sequences of chlorobium as well as Bacteroidesthetaiotaomicron and ppm gingiv., there is an additional domain justdownstream of the coding sequence that is unique to them;

(4) there is always a stem-loop in the coding sequence of theactinobacteria (Felden et al., 1999); and

(5) all the beta proteobacteria possess a sequence insertion inpseudoknot PK2 (shown in the alignment).

The novel sequences described herein, when aligned, show that specificstructural domains within tmRNA are strictly conserved, as for examplepseudoknot PK1 is located upstream (at the 5′-side) of the codingsequence. As previously disclosed, this pseudoknot is a target forfuture antibacterial drugs. Moreover, recent data have shown that thisPK1 pseudoknot, among all the four pseudoknots within tmRNA genesequences (sometimes there's only 2 or 3 detectable pseudoknots,depending upon the sequences), is the only one that its correct foldingis essential for the biological activity of tmRNA (Nameki et al., 1999;Nameki et al., 2000).

It has recently been discovered that even the alpha-proteobacteriapossess tmRNA genes. These genes are permuted and are made in two parts,connected via a processed linker. These tmRNA gene sequences fromalpha-proteobacteria were not found in the course of the presentinvention because usual PCR methods could not amplify them.

Recent reports have shown that whereas the gene encoding tmRNA isnon-essential in E. coli (does not kill the bacteria when disrupted), itis indeed essential in Neisseria gonorrheae (Huang et al., 2000). Also,tmRNA is directly involved in Salmonella typhymurium pathogenticity(Julio et al., 2000).

In summary, tmRNA genes are present in all eubacterial genomes, with noexceptions, but are not present in any genomes from archebacteries oreukaryotes, with the exception of some chloroplasts. The very specificlocation of tmRNA genes within one of the three main kingdoms of lifemake them ideal targets for the design of novel antibiotics that will,in principle, interfere very weakly with human biochemistry, compared tousual antibiotics. For a recent review about designing novelantibiotics, see Breithaupt (1999).

The present invention is also directed to diagnostic assays and kits forthe detection of bacterial infection, particularly infections caused bybacterial agents disclosed herein. In one embodiment, the codingsequence of each bacterial species is used to design specific primersfor use in amplification-based diagnostic assays for infectiousdiseases. Specific primers are designed in accordance with well knowntechniques, and such design is readily done by a skilled artisan.Amplification-based diagnostic assays are performed in accordance withconventional techniques well known to skilled artisans. Examples ofamplification-based assays include, but are not limited to, polymerasechain reaction (PCR) amplification, strand displacement amplification(SDA), ligase chain reaction (LCR) amplification, nucleic acid sequencebased amplification (3SR or NASBA) and amplification methods based onthe use of Q-beta replicase.

Drugs which target the sequences described herein are active agents canbe formulated in pharmaceutical compositions, which are preparedaccording to conventional pharmaceutical compounding techniques(Remington's, 1990). The composition may contain the active agent orpharmaceutically acceptable salts of the active agent. Thesecompositions may comprise, in addition to one of the active substances,a pharmaceutically acceptable excipient, carrier, buffer, stabilizer orother materials well known in the art. Such materials should benon-toxic and should not interfere with the efficacy of the activeingredient. The carrier may take a wide variety of forms depending onthe form of preparation desired for administration, e.g., intravenous,oral, intrathecal, epineural or parenteral.

For oral administration, the compounds can be formulated into solid orliquid preparations such as capsules, pills, tablets, lozenges, melts,powders, suspensions or emulsions. In preparing the compositions in oraldosage form, any of the usual pharmaceutical media may be employed, suchas, for example, water, glycols, oils, alcohols, flavoring agents,preservatives, coloring agents, suspending agents, and the like in thecase of oral liquid preparations (such as, for example, suspensions,elixirs and solutions); or carriers such as starches, sugars, diluents,granulating agents, lubricants, binders, disintegrating agents and thelike in the case of oral solid preparations (such as, for example,powders, capsules and tablets). Because of their ease in administration,tablets and capsules represent the most advantageous oral dosage unitform, in which case solid pharmaceutical carriers are obviouslyemployed. If desired, tablets may be sugar-coated or enteric-coated bystandard techniques. The active agent can be encapsulated to make itstable to passage through the gastrointestinal tract while at the sametime allowing for passage across the blood brain barrier. See forexample, WO 96/11698.

For parenteral administration, the compound may dissolved in apharmaceutical carrier and administered as either a solution of asuspension. Illustrative of suitable carriers are water, saline,dextrose solutions, fructose solutions, ethanol, or oils of animal,vegetative or synthetic origin. The carrier may also contain otheringredients, for example, preservatives, suspending agents, solubilizingagents, buffers and the like. When the compounds are being administeredintrathecally, they may also be dissolved in cerebrospinal fluid.

The active agent is preferably administered in an therapeuticallyeffective amount. The actual amount administered, and the rate andtime-course of administration, will depend on the nature and severity ofthe condition being treated. Prescription of treatment, e.g. decisionson dosage, timing, etc., is within the responsibility of generalpractitioners or specialists, and typically takes account of thedisorder to be treated, the condition of the individual patient, thesite of delivery, the method of administration and other factors knownto practitioners. Examples of techniques and protocols can be found inRemington's Pharmaceutical Sciences (18).

Alternatively, targeting therapies may be used to deliver the activeagent more specifically to certain types of cell, by the use oftargeting systems such as antibodies or cell-specific ligands. Targetingmay be desirable for a variety of reasons, e.g. if the agent isunacceptably toxic, or would otherwise require too high a dosage, orotherwise be unable to enter the target cells.

Antisense active agents can also be delivered by techniques described inU.S. Pat. Nos. 5,811,088; 5,861,290 and 5,767,102.

EXAMPLES

The present invention is further detailed in the following Examples,which are offered by way of illustration and are not intended to limitthe invention in any manner. Standard techniques well known in the artor the techniques specifically described below are utilized.

Example 1 Materials and Methods

1. Extraction of Genomic DNA

Bacterial genomic DNAs were prepared from ≈10 mg freeze-dried cellsprovided from ATCC (American Type Culture Collection, Virginia, USA).Cell pellets were resuspended in 750 μL of lysis buffer (50 mM Tris (pH8.0), 50 mM EDTA and 20% sucrose). 150 μL of a 10 mg/mL solution oflysozyme was mixed and let stand at room temperature for 15 min. 150 μLof 1% SDS was added and let stand at room temperature for 15 minutes.Four to five phenol/chloroform extractions were performed, until thesample was clear and there was no interphase. Two to five μL of a 10mg/mL solution of RNase DNase-free was added and incubated at roomtemperature for 30 minutes. After a phenol/chloroform extraction of theenzyme, the genomic DNA was precipitated with 1/10 volume of 3M NaOAc(pH 5.5) and 1 volume isopropanol, and stored at −20° C. for 2 hours.After centrifugation, the genomic DNAs were washed with 70% ethanol,vacuum-dried and diluted in sterile water to a final concentration of 10ng/μL.

2. Primer Sets for PCR Reactions

The following primer sets were used during the PCR:

primer set A (based on E. coli tmRNA termini): 5′-GGG GCT GAT TCT GGATTC GAC-3′ (SEQ ID NO: 1) and 5′-TGG AGC TGG CGG GAG TTG AAC-3′; (SEQ IDNO: 2) primer set B (based on T. neapolitana tmRNA termini): 5′-GGG GGCGGA AAG GAT TCG ACG-3′ (SEQ ID NO: 3) and 5′-TGG AGG CGG CGG GAA TCGAAC-3′; (SEQ ID NO: 4) primer set C (based on M. pneumoniae tmRNAtermini): 5′-GGG GAT GTC ATG GTT TTG ACA-3′ (SEQ ID NO: 5) and 5′-TGGAGA TGG CGG GAA TCG AAC-3′; (SEQ ID NO: 6) and primer set D (based on C.tepidum tmRNA termini): 5′-GGG GAT GAC AGG CTA TCG ACA-3′ (SEQ ID NO: 7)and 5′-TGG AGA TGG CGG GAC TTG AAC-3′. (SEQ ID NO: 8)

3. PCR Reaction

Sequences of tmRNA genes were obtained by polymerase chain reaction(PCR) in 25 μL using 40 ng of genomic DNA per reaction. The followinggeneral scheme was utilized for all of the sequences:

(a) 94° C. to 96° C. for 4 min. (first denaturation of genomic DNAs,done only once); then

(b) 35 to 40 PCR cycles with 2.5 to 5 Units of Taq DNA polymerase in a25 μL reaction volume, according to the following scheme (40 ng ofgenomic DNAs/PCR reaction):

-   -   1. denature at 94° to- 96° C. for 25 to 30 sec;    -   2. anneal at 44° to 55° C. for 20 to 30 sec; and    -   3. extension at 72° C. for 10 sec.        The magnesium conc. was optimized for each phyla from 3.5 to        13.5 mM.

4. Elution of Amplified DNAs

The various PCR-amplified tmDNA bands were gel purified (5% PAGE),stained (ethidium bromide staining), cut using a sterile razor blade,and shaken over-night (passive elution, using a vibrator) in a 350 μlsolution containing 10 mM Tris-HCl buffer (pH 8.1). The following day,the PCR amplified tmDNAs were ethanol precipitated, washed in 70% ETOH,vacuum dried and the DNA pellets were dissolved in 18 μl of RNase-DNasefree sterile water.

5. DNA Sequencing

Six μL of amplified DNAs were added to 3.2 picomoles of the primer thatwas used in the PCR. To verify the novel tmDNA sequences, each of thetwo primers were used independently to sequence each of the twoPCR-amplified DNA strands. Some tmDNAs were already engineered at their5′-ends with a T7 promoter, to be able to transcribe directly the tmDNAsinto tmRNAs by in vitro transcription.

Dye terminator sequencing was achieved at the DNA sequencing facility ofthe Human Genetics Institute. In addition to novel tmRNA sequences thatare not available publicly, several tmDNA sequences that were alreadyknown have been verified and several sequencing mistakes have been foundand corrected (especially for Alcaligenes eutrophus tmRNA).

Example 2 Amplification Reactions for Eubacterial tmDNA

Eubacterial tmDNA was amplified by PCR in accordance with Example 1,using the following conditions.

Acidobacterium:

Primer Set B; Annealing temp. during PCR: 53° C. for 20 sec; Mg²⁺ conc.:4.5 mM.

Coprothermobacter:

Primer Set B; Annealing temp. during PCR: 55° C. for 30 sec; Mg²⁺ conc.:5.5 mM.

Cytophagales:

Primer Set A; Annealing temp. during PCR: 46° C. for 30 sec; Mg²⁺ conc.:4.5 mM.

Dictyoglomus:

Primer set B; Annealing temp. during PCR: 55° C. for 30 sec; Mg²⁺ conc.:4.5 mM.

Environmental Samples:

Sludge DNA

Primer set C; Annealing temp. during PCR: 51° C. for 20 ^(sec; Mg2+)conc.: 13.5 mM.

Rumenal Fluid DNA

Primer set D; Annealing temp. during PCR: 50° C. for 30 sec; Mg²⁺ conc.:9.5 mM.

Fibrobacter:

Primer set A; Annealing temp. during PCR: 51° C.; Mg²⁺ conc.: 3.5 mM.

Firmicutes:

Fusobacteria:

Primer set A; Annealing temp. during PCR: 52° C.; Mg²⁺ conc.: 5.5 mM.

High G-C:

Primer set A; Annealing temp. during PCR: 50-55° C.; Mg²⁺ conc.: 4.5 mM.

Low G-C:

Primer sets A or B; Annealing temp. during PCR: 52° C.; Mg²⁺ conc.: 5.5to 7.5 mM.

Mycoplasmes:

Primer set A; Annealing temp. during PCR: 52° C.; Mg²⁺ conc.: 3.5 to 5.5mM.

Green Non-Sulfur:

Primer sets A or B; Annealing temp. during PCR: 46 to 52° C.; Mg²⁺conc.: 4.5 mM.

Green Sulfur:

Primer set A; Annealing temp. during PCR: 46° C.; Mg²⁺ conc.: 4.5 mM.

Planctomycetales:

Primer set A; Annealing temp. during PCR: 48 to 52° C.; Mg²⁺ conc.: 7.5mM.

Proteobacteria:

beta:

Primer sets A and/or B; Annealing temp. during PCR: 50° C. for 25 sec;Mg²⁺ conc.: 3.5 mM.

delta:

Primer set B; Annealing temp. during PCR: 55° C.; Mg²⁺ conc.: 3.5 to 4.5mM.

epsilon:

Primer set A; Annealing temp. during PCR: 46° C. for 30 sec; Mg²⁺ conc.:3.5 mM.

gamma:

Primer set A; Annealing temp. during PCR: 44 C for 30 sec; Mg²⁺ conc.:5.5 mM.

Spirochetes:

Primer set A; Annealing temp. during PCR: 52° C.; Mg²⁺ conc.: 4.5 mM.

Thermodesulfobacterium:

Primer set B; Annealing temp. during PCR: 55° C.; Mg²⁺ conc.: 5.5 mM.

Thermotogales:

Primer set B; Annealing temp. during PCR: 46° C.; Mg²⁺ conc.: 7.5 mM.

Deinococcales:

Primer set B; Annealing temp. during PCR: 52° C.; Mg²⁺ conc.: 3.5 mM.

Verrucomicrobia:

Primer set A; Annealing temp. during PCR: 53° C. for 25 sec; Mg²⁺ conc.:3.5 mM.

Example 3 Amplification of Eubacterial tmDNA

Specific PCR amplification of tmRNA genes was achieved for boththermophilic and mesophilic eubacterial tmRNA genes. For the novel tmDNAgenes found in thermophiles, both the magnesium concentration and theannealing temperature (FIG. 1A) were optimized. As shown in FIG. 1A, aspecific amplification of Thermus aquaticus tmDNA was observed with anannealing temperature around 50° C., whereas at higher temperaturesthere is a gradual decrease in the amount of amplified tmDNA. Formesophiles, the magnesium concentration during PCR was critical (FIG.1B), but the annealing temperature could vary from 44° C. to 60° C.without significant effects on the amplification. FIG. 1B shows variouseffects of increasing the magnesium concentration on the PCRamplification of three novel eubacterial tmDNA genes. Increasingmagnesium concentration from 3.5 mM to 5.5 mM has either a negative(FIG. 1B, panel 1), a positive (FIG. 1B, panel 2) or no effect onspecifically amplifying eubacterial tmDNA genes.

According to these procedures, tmRNA genes from many eubacteriaincluding known human pathogens were amplified. The PCR was facilitatedby sequence conservation at both 5′ and 3′ ends and was performed asdescribed (Williams and Bartel, 1996), with modifications. This studywas initiated to collect further sequences from eubacterial tmDNA genes,as well as to test experimentally whether tmDNA genes could be found inall bacterial phyla or subgroups. 51 new tmDNA sequences were determined(FIG. 2), including sequences from members of 8 additional phyla and 1subgroup (shaded boxes in FIG. 2). The 58 new tmDNA sequences are setforth in Tables 1-58. This brings coverage to a total of 104 sequencesin 19 bacterial phyla. Interestingly, tmDNA sequences could be amplifiedfrom all species tested apart from those in the alpha-Proteobacteria.Five genomic DNAs from alpha-Proteobacteria (Agrobacterium tumefaciens,Bartonella henselae, Bartonella quintana, Rhodospirillum rubrum andRickettsia prowazekii) were extensively checked using variousoligonucleotides, annealing temperatures and magnesium concentrations.No specific amplified tmDNA sequences were detected in this subgroup.Moreover, no putative tmDNA sequences could be identified (resultsherein and Williams, 1999) by Blast searches on the 1 fully sequenced(Rickettsia prowazekii) and 2 nearly completed (Caulobacter crescentusand Rhodobacter capsulatus) alpha-proteobacterial genomes (FIG. 2).

It cannot be ruled out that tmDNA sequences may have largely diverged inthe alpha-proteobacterial sub-group compared to other bacterial phyla,and that both PCR methods and Blast searches are missing the relevantsequences. While tmRNA is dispensable in E. coli (Ando et al., 1996), itis striking that it has been found in all bacteria tested other than thealpha-Proteobacteria. The alpha-Proteobacteria have undergone reductiveevolution. This has been more intensive in one of the two sub-classesthan in the other (Gray and Spencer, 1996), but tmRNA sequences have notbeen found even in the sub-class with the larger genome. Based onsequence comparison, the alpha-Proteobacteria and mitochondria areevolutionary relatives (Yang et al., 1985; Andersson et al., 1998). Thedrastic downsizing in what has become mitochondrial genomes means thatit is not reasonable to draw inferences on the relationship betweenalpha-Proteobacteria and mitochondria based on their mutual apparentabsence of tmRNA. It is nevertheless, of interest, that at least somechloroplasts and cyanelle genomes have tmDNA sequences, and thecyanobacteria, with which they are evolutionary related, also havetmRNA.

TABLE 1 tmDNA Sequence for Acidobacterium capsulatum (Acidobacterium)GGGGGCGGAAAGGATTCGACGGGGTTGACTGCGGCAAAGAGGCATGCCGGGGGGTGGGCACCCG (SEQ IDNO: 9) TAATCGCTCGCAAAACAATACTTGCCAACAACAATCTGGCACTCGCAGCTTAATTAAATAAGTTGCCGTCCTCTGAGGCTTCGCCTGTGGGCCGAGGCAGGACGTCATACAGCAGGCTGGTTCCTTCGGCTGGGTCTGGGCCGCGGGGATGAGATCCACGGACTAGCATTCTGCGTATCTTGTCGCTTCTAAGCGCAGAGTGCGAAACCTAAAGGAATGCGACTGAGCATGGAGTCTCTTTTCTGACACCAATTTCGGACGCGGGTTCGATTCCCGCCGCCTCCACCA

TABLE 2 tmDNA Sequence for Coprothermobacter proteolyticus (60 degrees)GGGGGCGGAAAGGATTCGACGGGGAGTCGGAGCCTTGAGCTGCAGGCAGGGTTGGCTGCCACAC (SEQ IDNO: 10) CTTAAAAAGGGTAGCAAGGCAAAAATAAATGCCGAACCAGAATTTGCACTAGCTGCTTAATGTAAGCAGCCGCTCTCCAAACTGAGGCTGCATAAGTTTGGAAGAGCGTCAACCCATGCAGCGGCTCTTAAGCAGTGGCACCAGCTGTTTAAGGGTGAAAAGAGTGGTGCTGGGCAGTGCGGTTGGGCTTCCTGGGCTGCACTGTCGAGACTTCACAGGAGGGCTAAGCCTGTAGACGCGAAAGGTGGCGGCTCGTCGGACGCGGGTTCGATTCCCGCCGCCTCCACCA

TABLE 3 tmDNA Sequence for Bacteroides thetaiotaomicron(bacteroides/flavobacterium)GGGGCTGATTCTGGATTCGACAGCGGGCAGAAATGGTAGGTAAGCATGCAGTGGGTCGGTAATT (SEQ IDNO: 11) TCCACTTAAATCTCAGTTATCAAAACTTTATCTGGCGAAACTAATTACGCTCTTGCTGCTTAATCGAATCACAGTAGATTAGCTTAATCCAGGCACTAGGTGCCAGGACGAGACATCACTCGGAAGCTGTTGCTCCGAAGCATTCCGGTTCAGTGGTGCAGTAACATCGGGGATAGTCAGAAGCGGCCTCGCGTTTTTGATGAAACTTTAGAGGATAAGGCAGGAATTGATGGCTTTGGTTCTGCTCCTGCACGAAAATTTAGGCAAAGATAAGCATGTAGAAAGCTTATGATTTCCTCGTTTGGACGAGGGTTCAACTCCCGCCAGCTCCACCA

TABLE 4 tmDNA Sequence for Dictyoglomus thermophilum (70 degrees)GGGGCTGATTCTGGATTCGACAGGGAGTACAAGGATCAAAAGCTGCAAGCCGAGGTGCCGTTAC (SEQ IDNO: 12) CTCGTAAAACAACGGCAAAAAAGAAGTGCCAACACAAATTTAGCATTAGCTGCTTAATTTAGCAGCTACGCTCTTCTAACCCGGGCTGGCAGGGTTAGAAGGGTGTCATAATGAGCCAGCTGCCCCTTCCGACTCCCCTAAGGAAGGGAAAGATGTAGGGGATAGGTGCTTACAGAATCCTGCGGGAGGGAGTCTGTAAGTGCCGAAAAGTTAAAACTCCCGCTAAGCTTGTAGAGGCTTTTGATTCTTGCTCTCTGGACGCGGGTTCAACTCCCGCCAGCTCCACCA

TABLE 5 tmDNA Sequence for Environmental Sample from Rumenal FluidACGCCCTTGTCTCAGACGAGGGCACTCGTTAAAAAGTCTGAAAAGAATAACTGCAGAACCTGTA (SEQ IDNO: 13) GCTATGGCTGCTTAATTTAAGGGCAACCCTTGGATCCGCCTCCATCCCGAAGGGGTGGCATCCGAGTCGCAAATCGGGATAGGATGGATCTTGGCAACGAGGAGTACATCCGAAATTTGTCGCTGCTGGCTGAAGCATCGCCGTTCCTCTTTGGGCGTGGCAAGGCAAGATTAAATTCAGAGGATAAGCGTGTAGTAGCGAGTGAGTAGGTGTTTTTGGACGCGGGTTCAAGTCCCGCCATCTCCACCA

TABLE 6 tmDNA Sequence for Environmental Sample from SludgeGGGGATGTCATGGTTTTGACAGGGAACCAGGAGGTGTGAGATGCATGCCGGAGACGCTGTCCGC (SEQ IDNO: 14) TCCGTTATCAAGCAGCAAACAAAACTAATTGCAAACAACAATTACTCCTTAGCAGCGTAAGCAGCTAACGTTCAACCTCTCCGGACCGCCGGGAGGGGATTTGGGCGTCGAAACAGCGCGGACGCTCCGGATAGGACGCCCATAATATCCGGCTAAGACCATGGGTCTGGCTCTCGCGGGTCTGATTGTCTTCCACCGCGCGGGCCGCGATCAAAGACAACTAAGCATGTAGGTTCTTGCATGGCCTGTTCTTTGGACGCGGGTTCGATTCCCGCCATCTCCACCA

TABLE 7 tmDNA Sequence for Fibrobacter succinogenes (Fibrobacter)GGGGCTGATTCTGGATTCGACAGGGTTACCGAAGTGTTAGTTGCAAGTCGAGGTCTCAGACGAG (SEQ IDNO: 15) GGCTACTCGTTAAAAAGTCTGAAAAAAAATAAGTGCTGACGAAAACTACGCACTCGCTGCCTAATTAACGGCAACGCCGGGCCTCATTCCGCTCCCATCGGGGTGTACGTCCGGACGCAATATGGGATAGGGAAGTGTCATGCCTGGGGGCATCTCCCGAGATTTTCTAGGCTGGTCAAACTCCGCGCCGACCTTCTTGGGCGTGGATAAGACGAGATCTTAAATTCGAAGGGAACACTTGTAGGAACGTACATGGACGTGATTTTGGACAGGGGTTCAACTCCCGCCAGCTCCA

TABLE 8 tmDNA Sequence for Fusobacterium mortiferumGGGGCTGATTCTGGATTCGACGGGGTTATGAGGTTATAGGTAGCATGCCAGGATGACCGCTGTG (SEQ IDNO: 16) AGAGGTCAACACATCGTTTAGATGGAAACAGAAATTACGCTTTAGCTGCTTAATTAGTCAGCTCACCTCTGGTTTCTCTCTTCTGTAGGAGAATCCAACCGAGGTGTTACCAATATACAGATTACCTTTAGTGATTTCTCTAAGCTCAAAGGGACATTTTAGAGAATAGCTTCAGTTAGCCCTGTCTGCGGGAGTGATTGTTGCGAAATAAAATAGTAGACTAAGCATTGTAGAAGCCTATGGCGCTGGTAGTTTCGGACACGGGTTCAACTCCCGCCAGCTCCAA

TABLE 9 tmDNA Sequence for Corynebacterium xerosis (gram +, high G-Ccontent)GGGGCTGATTCTGGATTCGACTTCGTACATTGAGCCAGGGGAAGCGTGCCGGTGAAGGCTGGAG (SEQ IDNO: 17) ACCACCGCAAGCGTCGCAGCAACCAATTAAGCGCCGAGAACTCTCAGCGCGACTACGCCCTCGCTGCCTAAGCAGCGACCGCGTGTCTGTCAGACCGGGTAGGCCTCTGATCCGGACCCTGGCATCGTTTAGTGGGGCTCGCTCGCCGACTTGGTCGCAAGGGTCGGCGGGGACACTCACTTGCGACTGGGCCCGTCATCCGGTCATGTTCGACTGAACCGGAGGGCCGAGCAGAGACCACGCGCGAACTGCGCACGGAGAAGCCCTGGCGAGGTGACGGAGGACCCGGGTTCAACTCCCGCCAGCTCCACCA

TABLE 10 tmDNA Sequence for Micrococcus luteus (parfait)GGGGCTATTCTGGATTCGACGGTGTGTGTCGCGTCGGGAGAAGCGGGCCGAGGATGCAGAGTCA (SEQ IDNO: 18) TCTCGTCAAACGCTCTCTGCAAACCAATAAGTGCCGAATCCAAGCGCACTGACTTCGCTCTCGCTGCCTGATCAGTGATCGAGTCCGTCACCCCGAGGTCGCTGTCGCCTCGGATCGTGGCGTCAGCTAGATAGCCACTGGGCGTCACCCTCGCCGGGGGTCGTGACGCCGACATCAATCCGGCTGGGTCCGGGTTGGCCGCCCGTCTGCGGGACGGCCAGGACCGAGCAACACCCACAGCAGACTGCGCCCGGAGAAGACCTGGCAACACCTCATCGGACGCGGGTTCAACTCCCGCANTCCCACCA

TABLE 11 tmDNA Sequence for Mycobacterium smegmatisTCATCTCGGCTTGTTCGCGTGACCGGGAGATCCGAGTAGAGACATAGCGAACTGCGCACGGAGA (SEQ IDNO: 19) GGGGCTGATTCCTGGATTCGACTTCGAGCATCGAATCCAGGGAAGCGTGCCGGTGCAGGCAAGAGACCACCGTAAGCGTCGTTGCAACCAATTAAGCGCCGATTCCAATCAGCGCGACTACGCCCTCGCTGCCTAAGCGACGGCTGGTCTGTCAGACCGGGAGTGCCCTCGGCCCGGATCCTGGCATCAGCTAGAGGGACCCACCCACGGGTTCGGTCGCGGGACCTGTGGGGACATCAAACAGCGACTGGGATCGAGCCTCGAGGACATGCCGTAGGACCCGGGTTCAACTCCCGCCAGCTCCACCA

TABLE 12 tmDNA Sequence for Bacillus badiusGGGGGTGATTCTGGATTCGACAGGGATAGTTCGAGCTTGGGCTGCGAGCCGGAGGGCCGTCTTC (SEQ IDNO: 20) GTACCAACGCAAACGCCTAAATATAACTGGCAAAAAAGATTTAGCTTTAGCTGCCTAATATAGGTTCAGCTGCTCCTCCCGCTATCGTCCATGTAGTCGGGTAAGGGGTCCAAACTTAGTGGACTACGCCGGAGTTCTCCGCCTGGGGACAAAGGAAGAGATCAATCAGGCTAGCTGCCCGGACGCCCGTCGATAGGCAAAAGGAACAGTGAACCCCAAATATATCGACTACGCTCGTAGACGTTCAAGTGGCGTTATCTTTGGACGTGGGTTCAACTCCCGCCAGCTCCA

TABLE 13 tmDNA Sequence for Bacillus brevisGGGGGCGGAAAGGATTCGACGGGGATGGTAGAGCATGAGAAGCGAGCCGGGGGGTTGCGGACCT (SEQ IDNO: 21) CGTCACCAACGCAAACGCCATTAACTGGCAACAAACAACTTTCTCTCGCTGCTTAATAACCAGTGAGGCTCTCCCACTGCATCGGCCCGTGTGCCGTGGATAGGGCTCAACTTTAACGGGCTACGCCGGAGGCTTCCGCCTGGAGCCAAAGGAAGAAGACCAATCAGGCTAGGTGCCAGGTCAGCGCGTCACTCCGCGAATCTGTCACCGAAACTCTAAACGAGTGACTGCGCTCGGAGATGCTCATGTATCGCTGTTTTCGGACGGGGGTTCGATTCCCGCCGCCTCACCCA

TABLE 14 tmDNA Sequence for Bacillus thermoleovorans (50-60 degres)GGGGGCGGAAAGGATTCGACGGGGGTAGGTCGAGCTTAAGCGGCGAGCCGAGGGGGACGTCCTC (SEQ IDNO: 22) GTAAAAACGTCACCTAAAGATAACTGGCAAACAAAACTACGCTTTAGCTGCCTAATTGCTGCAGCTAGCTCCTCCCGCCATCGCCCGCGTGGCGTTCGAGGGGCTCATATGGAGCGGGCTACGCCCAAATCCGCCGCCTGAGGATGAGGGAAGAGACGAATCAGGCTAGCCGCCGGGAGGCCTGTCGGTAGGCGGAACGGACGGCGAAGCGAAATATACCGACTACGCTCGTAGATGCTTAAGTGGCGATGCCTCTGGACGTGGGTTCGATTCCCGCCGCCTCCCCACCA

TABLE 15 tmDNA Sequence for Clostridium innocuumGGGGGCGGAAAGGATTCGACGGGGATATGTCTGGTACAGACTGCAGTCGAGTGGTTACGTAATA (SEQ IDNO: 23) ACCAATTAAATTTAAACGGAAAAACTAAATTAGCTAACCTCTTTGGTGGAAACCAGAGAATGGCTTTCGCTGCTTAATAACCGATATAGGTTCGCAGCCGCCTCTGCATGCTTCTTCCTTGACCATGTGGATGTGCGCGTAAGACGCAAGGGATAAGGAATCTGGTTTGCCTGAGATCAGATTCACGAAAATTCTTCAGGCACATTCATCAGCGGATGTTCATGACCTGCTGATGTCTTAATCTTCATGGACTAAACTGTAGAGGTCTGTACGTGGGGCTGTTTCTGGACAGGAGTTCGATTCCCGCCGCCTCACCACCA

TABLE 16 tmDNA Sequence for Clostridium lentocellumGGGGGCGGAAAGGATTCGACGGGGGTCACATCTACTGGGGCAGCCATCCGTAGAACGCCGGAGT (SEQ IDNO: 24) CTACGTTAAAAGCTGGCACTTAAAGTAAACGCTGAAGATAATTTAGCAATCGCTGCCTAATTAAGGCGCAGTCCTCCTAGGTCTTCCGCAGCCTAGATCAGGGCTTCGACTCGCGGATCCTTCACCTGGCAAAGCTTTGAGCCAACGTGAACACTATGAAGCTACTAAAATCTAGAGCCTGTCTTTGGGCGCTAGATGGAGGGAATGTCAAAACAAAGAATATGATGGTAGAGACCACGCTATATGGGCTTTCGGACAGGGGTTCGATTCCCGCCGCCTTCACCA

TABLE 17 tmDNA Sequence for Clostridium perfringensGGGGCTGATTCTGGATTCGACGGGGGTAAGATGGGTTTGATAAGCGAGTCGAGGGAAGCATGGT (SEQ IDNO: 25) GCCTCGATAATAAAGTATGCATTAAAGATAAACGCAGAAGATAATTTTGCATTAGCAGCTTAATTTAGCGCTGCTCATCCTTCCTCAATTGCCCACGGTTGAGAGTAAGGGTGTCATTTAAAAGTGGGGAACCGAGCCTAGCAAAGCTTTGAGCTAGGAACGGAATTTATGAAGCTTACCAAAGAGGAAGTTTGTCTGTGGACGTTCTCTGAGGGAATTTTAAAACACAAGACTACACTCGTAGAAAGTCTTACTGGTCTGCTTTCGGACACGGGTTCAACTCCCGCCACTCCA

TABLE 18 tmDNA Sequence for Clostridium stercorariumGGGGGCGGAAAGGATTCGACGGGGTTATTGAAGCAAGAGTAGCGGGTAGAGGATTCTCGTTGGC (SEQ IDNO: 26) CTCTTTAAAAAACGAGAGCTAAAAATAAACGCAAACAACGATAACTACGCTTTAGCTGCTGCGTAAGTAACACGCAGCCCGTCGGCCCCGGGGTTCCTGCGCCTCGGGATACCGGCGTCATCAAGGCAGGGAACCAGCCGGATCAGGCTTCAGGTCCGGTGGGATTTAATGAAGCTACCGACTTATAAAGCCTGTCTCTGGGCGTTATAAGAAGGGAATGTCAAAACAGAGACTGCACCCGGAGAAGCTCTTGTGGATATGGTTCCGGACACGAGTTCGATTCCCGCCGCCTCCACCA

TABLE 19 tmDNA Sequence for Enterococcus faecium (sp.)GGGGCTGATTATGGATTCGACAGGATNGTTGAGCTTGAATTGCGTTTCGTAGGTTACGGCTACG (SEQ IDNO: 27) TTAAAACGTTACAGTTAAATATAACTGCTAAAAACGAAAACAATTCTTTCGCTTTAGCTGCCTAAAAACCAGCTAGCGAAGATCCTCCCGGCATCGCCCATGTGCTCGGGTCAGGGTCCTAATCGAAGTGGGATACGCTAAATTTTTCCGTCTGTAAAATTTAGAGGAGCTTACCAGACTAGCAATACAAGAATGCCTGTCACTCGGCACGCTGTAAAGCGAACCTTTAAATGAGTGTCTATGAACGTAGAGATTTAAGTGGGAATATGTTTTGGACGCGGGTTCAACTCCCGCCAGCTCCACCA

TABLE 20 tmDNA Sequence for Heliobacillus mobilis (photosyn/gram +)GGGGCTGATTCTGGATTCGACGGGGAACGTGTTTGCTTGGGATGCGAGCCGGGTTGCCGCCAGG (SEQ IDNO: 28) ACCGTAAAAAGGGCGGAAGGCTTTAATTGCCGAAGATAACTACGCTTTAGCTGCTTAATTGCAGTCTAACCTCTTCTCCTCTGTGCTCTCGGTGAGGATGTAAGGGGTCATTTAAGAGAGCTGGCTTCGACCAATTCTCGGAGGTCCAAGCGAGATTTATCGAGATAGCCTGACCAACGCTCTGTCTGCCGTGCGGAAGGAAGGCGAAATCTAAAACGACAGACTACGCTCGTAGTGTCCTTTGTGGGCATTTCTTCGGACGCGGGTTCAACTCCCGCCAGCTCCACCA

TABLE 21 tmDNA Sequence for Heliospirillum gestiiGGGGCTGATTCTGGATTCGACGGGGAACGTGTTTGCTTAGGACGCGAGCCGGGTTGCCGCCAGG (SEQ IDNO: 29) ACCGTAAAAAGGGCGGAAGGCTTTAATTGCCGAAGATAACTACGCTTTAGCTGCTTAATTGCAGTCTAACCTCTTCTCCTCTGTGCTCTCGGTGAGGATGTAAGGGGTCATTTAAGAGAGCTGGCTCGAACCAATTCTCGGAGGTTCGGGTAAGACTTATCGAGATAGCCTGACCAACGCTCTGTCTGCCGTGCGGAAGGATGGCGAAATCTAAAACGACAGAATACGCTCGTAGTGTCCTTTGTGGGCATTTCTTCGGACGCGGGTTCAACTCCCGCCAGCTCCACCA

TABLE 22 tmDNA Sequence for Lactobacillus acidophilusGGGGCTGATTCTGGATTCGACAGGCGTAGACCCGCATTGACTGCGGTTCGTAGGTTACGTCTAC (SEQ IDNO: 30) GTAAAAACGTTACAGTTAAATATAACTGCAAATAACAAAAATTCTTACGCATTAGCTGCTTAATTTAGCGCATGCGTTGCTCTTTGTCGGTTTACTCGTGGCTGACACTGAGTATCAACTTAGCGAGTTACGTTTAACTACCTCACCTGAATAGTTGAAAAGAGTCTTAGCAGGTTAGCTAGTCCATACTAGCCCTGTTATATGGCGTTTTGGACTAGTGAAGTTCAAGTAATATAACTATGATCGTAGAGGTCAGTGACGAGATGCGTTTGGACAGCGGGTTCAACTCCCGCCAGCTCCACCA

TABLE 23 tmDNA Sequence for Staphylococcus epidermidisGGGGCTGATTCTGCATTCGACAGGGGTCCCCGAGCTTATTAAGCGTGTGGAGGGTTGGCTCCGT (SEQ IDNO: 31) CATCAACACATTTCGGTTAAATATAACTGACAAATCAAACAATAATTTCGCAGTAGCTGCGTAATAGCCACTGCATCGCCTAACAGCATCTCCTACGTGCTGTTAACGCGATTCAACCCTAGTAGGATATGCTAAACACTGCCGCTTGAAGTCTGTTTAGATGAAATATAATCAAGCTAGTATCATGTTGGTTGTTTATTGCTTAGCATGATGCGAAAATTATCAATAAACTACACACGTAGAAAGATTTGTATCAGGACCTCTGGACGCGGGTTCAACTCCCGCCAGCTCCACCA

TABLE 24 tmDNA Sequence for Streptococcus faeciumGGGGCTGATTCTGGATTCGACAGGCACAGTTTGAGCTTGAATTGCGTTTCGTAGGTTACGTCTA (SEQ IDNO: 32) CGTTAAAACGTTACAGTTAAATATAACTGCTAAAAACGAAAACAACTCTTACGCTTTAGCTGCCTAAAAACAGTTAGCGTAGATCCTCTCGGCATCGCCCATGTGCTCGAGTAAGGGTCTCAAATTTAGTGGGATACGTGACAACTTTCCGTCTGTAAGTTGTTAAAGAGATCATCAGACTAGCGATACAGAATGCCTGTCACTCGGCAAGCTGTAAAGCGAAACCACAAATGAGTTGACTATGAACGTAGATTTTTAAGTGGCGATGTGTTTGGACGCGGGTTCAACTCCCGCCGTTCCACCA

TABLE 25 tmDNA Sequence for Thermoanaerobacterium saccharolyticum(Bacillus/clostridium)GGGGTAGTAGAGGTAAAAGTAGCGAGCCGAGGTTCCATCTGCTCGTAAAACGGTGGACTTAAAT (SEQ IDNO: 33) ATAAACGCAAACGATAATTTAGCTTACGCTGCTTAATTACAAGCAGCCGTTCAACCTTTGATTCCCACATCAAAGGATTGGGCGTCGATTTAGTGGGGAACTGATTTATCAAAGCTTTGAGATAAATCGGATTTTATGAAGCTACCAAAGCAGTTATCCTGTCACTGGGAGAACTGCAGAGGGAATGTCAAAACAGTGACTGCGCTCGGAGAAGCTTTTACTGTGACACCTTCGGACCGGGGTTCAACTCCCGCCAGCTCCACCA

TABLE 26 tmDNA Sequence for Mycoplasma fermentansGGGGCTGATTCTGGATTCGACATGCATTGGGTGATACTAATATCAGTAGTTTGGCAGACTATAA (SEQ IDNO: 34) TGCATCTAGGCTTTATAATCGCAGAAGATAAAAAAGCAGAAGAAGTTAATATTTCTTCACTTATGATTGCACAAAAAATGCAATCACAATCAAACCTTGCTTTCGCTTAGTTAAAAGTGACAAGTGGTTTTAAAGTTGACATTTTCCTATATATTTTAAAATCGGCTTTTAAGGAGAACAGGAGTCTGAAAGGGTTCCAAAAATCTATATTGTTTGCATTTCGGTAGTATAGATTAATTAGAAATGATAAACTGTAAAAAGTATTGGTATTGACTTGGTGTGTGGACTCGGGTTCAACTCCCGCCAGCTCCACCA

TABLE 27 tmDNA Sequence for Mycoplasma hyorhinisGGGGCTGATTCTGGATTCGACATACATAAAAGGATATAAATTGCAGTGGTCTTGTAAACCATAA (SEQ IDNO: 35) GACAATTTCTTTACTAAGCGGAAAAGAAAACAAAAAAGAAGATTATTCATTATTAATGAATGCTTCAACTCAATCAAATCTAGCTTTTGCATTTTAAAAAACTAGTAGACCAATTTGCTTCTCACGAATTGTAATCTTTATATTAGAGAATAGTTAAAAATCTGATCACTTTTTAATGAATTTATAGATCACAGGCTTTTTTAATCTTTTTGTTATTTTAGATAAAGAGTCTTCTTAAAAATAACTAAACTGTAGGAATTTATATTTAATTATGCGTGGACCCGGGTTCAACTCCCGCCAGCTCCACCA

TABLE 28 tmDNA Sequence for Mycoplasma pirumGGGGAGTCATGGTTTTGACATGAATGATGGACCCATAGAGGCAGTGGGGTATGCCCCTTATAGC (SEQ IDNO: 36) TCAAGGTTTAAATTAACCGACAAAACTGACGAAAACGTTGCCGTTGATACAAATTTATTAATCAACCAACAAGCTCAATTTAACTACGCATTTGCATAGTATAAAAAAATAAATTGTGCTACTCATTGTAATTAGGTTACTAAATTACTTTGTTTTATATAGTCCTGTAACTAGTTCTAGTGATGTCTATAAACTAGAATGAGATTTATAGACTTATTTGTTGGCGGTTGTGCCATAGCCTAAATCAACAAAGACAATTTATTTATGGTACTAAACTGTAGATTCTATGATGAAATTATTTGTGGAAACGGGTTCGATTCCCGCCATCTCCACCA

TABLE 29 tmDNA Sequence for Mycoplasma salivariumGGGGCTGATTCTGGATTCGACAGGCATTCGATTCATTATGTTGCAGTGGTTTGCAAACCATAAG (SEQ IDNO: 37) GCACTAGGCTTTTTTAAACGCAAAAGACCAAAAAACAGAAGATCAAGCAGTTGATCTAGCATTTATGAATAATTCACAAATGCAATCAAATCTAGTTTTCGCTTAGTAAAATTAGTCAATTTATTATGGTGCTCAACATAATAAATGGTAGTATGAGCTTAATATCATATGATTTTAGTTAATATGATAGGATTTGTAACTAAACTATGTTATAGAAATTTGTAAATTATATATATGACATAGGAAATTTAATTTACTAAACTGTAGATGCATAATGTTGAAGATGTGTGGACCGGGGTTCAACTCCCGCCAGCTCCACCA

TABLE 30 tmDNA Sequence for Herpetosiphon aurantiacusGGGGGCGGAAAGGATTCGACGGGGAGGGCCAATCGTAAGTGGCAAGCCGAGACGCTGAGCCTCG (SEQ IDNO: 38) TTAAATCGGCAACGCCATTAACTGGCAAAAACACTTTCCGCGCTCCTGTAGCGCTTGCTGCCTAATTAAGGCAACACGTCTCTACTAGCCTCAGCCCGATGGGCTTGTAGCGGCGACACTTAGTCGGGTCGCTCCCCTAGTTATGTCTGTGGGCTAGGGGCTAAGATTAACAGGCTGGTCGTGGCCCGCTTTGTCTATCGGGTGGTGCACCGATAAGATTTAATCAATAGACTACGCTTGTAGATGCTTGCGGTTTAACTTTTTGGACGCGGGTTCGATTCCCGCCGCCTCACCACCA

TABLE 31 tmDNA Sequence for Thermomicrobium roseum (352 nts, temp. 70degrees, green non sulfur)GGGGCTGATTCTGGATTCGACAGGGCCGTAGGTGCGAGGATTGCAGGTCGAGGTCGCCCACGAA (SEQ IDNO: 39) CTCGTAAAAAGGGGCAGCCAAGTAACTGGCGAGCGCGAACTCGCTCTGGCTGCGTAATTCACGCAGCCACGTCTGCCCGGACCCTTCCCTGGTGGGTTCGGAGCGGGCGCCGCAAGACCGGGGTGCCCCTGGCCCAAGCGCCGGTGCGGGCCAGGTCAAGCGTGATCCGGCTCGGCTGACCGGGATCCTGTCGGTGGGAGCCTGGCAGCGACAGTAGAACACCGACTAAGCCTGTAGCATATCCTCGGCTGAACGCTCTGGACGCGGGTTCAACTCCCGCCAGCTCCACCA

TABLE 32 tmDNA Sequence for Chlorobium limicolaGGGGCTGATTCTGGATTCGACAGGATACGTGTGAGATGTCGTTGCACTCCGAGTTTCAGCATGG (SEQ IDNO: 40) ACGGACTCGTTAAACAAGTCTATGTACCATTAGATGCAGACGATTATTCGTATGCAATGGCTGCCTGATTAGCACAAGTTAACTCAGACGCCATCGTCCTGCGGTGAATGCGCTTACTCTGAAGCCGCCGGATGGCATAACCCGCGCTTGAGCCTACGGGTTCGCGCAAGTAAGCTCCGTACATTCATGCCCGAGGGGCTGTGCGGGTAATTTCTCGGGATAAGGGGACGAACGCTGCTGGCGGTGTAATCGGCCCACGAAAACCCAATCACCAGAGATGAGTGTGGTGACTGCATCGAGCAGTGTTTTGGACGCGGGTTCAACTCCCGCCAGCTCCACCA

TABLE 33 tmDNA Sequence for Pirellula staleyi (planctomyces)GGGGCTGATTCTGGATTCGACCGGATAGCCTGAAGCGAATACGGCGTGCCGTGGTTGATCAGAT (SEQ IDNO: 41) GGCCACGTAAAAAGCTGATCACAAACTTAACTGCCGAGAGCAATCTCGCACTTGCTGCCTAACTAAACGGTAGCTTCCGACTGAGGGCTTTAGCCGGAGAGGCCCAAAAGTTGGTCACCAAATCCGGACCGCCTCGTGCCATGATCGAAACGCACGAGGTCAAAAAAGTTTCGATCTAGTGCAGGGTGTAGCCAGCAGCTAGGCGACAAACTGTGCAAAAATCAAATTTTCTGCTACGCACGTAGATGTGTTCGTGAAAATGTCTCGGGACGGGGGTTCAACTCCCGCCACTCCACCA

TABLE 34 tmDNA Sequence for Planctomyces limnophilusGGGGCTGATTCTGGATTCGACAACCTCTCAAGAGGAGCGTGGCCACTATGGGACTCGATTATGT (SEQ IDNO: 42) TGAATTCGTCATGGATCTTGAAGAGACCTTCGACATCAAACTGGATGACAAACATTTTTCAGCAGTCAAAACACCACGCGATTTGGCAATCATTATTCGGGATCAATTAGCTGCTGAAGGCAGAATCTGGGATGAATCGAATGCTTTTCGCAAAATCTCGAATTTGAATTGGACGATGTTGCCCGAGTTCCGGATGTGGACTCAAATCAAAAGCTCTCTACCAGTTTCTTTTCACCGACTGCGTCCCAGCACCCGTCTCGTTCAACTCCCGCCANTCCACCA

TABLE 35 tmDNA Sequence for Planctomyces marisGGGGCTGATTCTGGATTCGACTGGTTCACCGTATGTTAAGGTGGCGGTGCCGTGGTTGATCAGT (SEQ IDNO: 43) TGGCCACGTAAAAAGCTGATCACAATCTAATTGCAAACAAGCAATTTTCAATGGCTGCTTAATAAAAGCAACCCCGGCTTAGGAATCTCTGTCTGAGGAGTCCGACAGCTGGTCACAAAATCAGACTGGTATCAGATCAATGTCCGCTCCGTCTGATACGAGATTCGTGGTGGACTGGTTTCCAACAGGCTCTGTTTATCGTGCCCGAAGAAACGAGACTCAAACGATAAAATATGCACCGTAGAGGCTTTAGCTGAGGGTTCACAGGACGCGGGTTCAACTCCCGCCAGCTCCACCA

TABLE 36 tmDNA Sequence for Alcaligenes eutrophusGGGGTTGATTCTGGATTCGACGTGGGTTACAAAGCAGTGGAGGGCATACCGAGGACCCGTCACC (SEQ IDNO: 44) TCGTTAATCAATGGGAATGCAATAACTGCTAACGACGAACGTTACGCACTGGCCGCTTAATTGCGGCCGTCCTCGCACTGGCTCGCTGACGGGCTAGGGTCGCAAGACCACGCGAGGTCATTTACGTCAGATAAGCTCCGGAAGGGTCACGAAGCCGGGGACGAAAACCTAGTGACTCGCCGTCGTAGAGCGTGTTCGTCCGCGATGCGCCGGTTAAATCAAATGACAGAACTAAGTATGTAGAACTCTCTGTGGAGGGCTTACGGACGCGGGTTCAACTCCCGCCAGCTCCACCA

TABLE 37 tmDNA Sequence for Alcaligenes faecalis (beta proteobacteria)GGGGGCGGAAAGGATTCGACGGGGGTCAAGAAGCAGCACAGGGCGTGTCGAGCACCAGTACGCT (SEQ IDNO: 45) CGTAAATCCACTGGAAAACTATAAACGCCAACGACGAGCGTTTCGCTCTAGCCGCTTAAGGCTGGGCCACTGCACTAATTTGTCTTTGGGTTAGGTAGGGCAACCTACAGCAGTGTTATTTACAAAGAATCGAATCGGTCTGCGCCACGAAGTCCGGTTCTAAAACTTAGTGGATCGCCAAGGAAAGGCCTGTCAATTGGCATAGTCCAAGGTTAAAACTTAAAATTAATTGACTACACATGTAGAACTGTCTGTGGACGGCTTGCGGACGGGGGTTCGATTCCCGCCGCCTCCACCA

TABLE 38 tmDNA Sequence for Chromobacterium violaceum (beta-purple)GGGGCTGATTCTGGATTCGACGGGGGTTGCGAAGCAGATGAGGGCATACCGGGATTTCAGTCAC (SEQ IDNO: 46) CCCGTAAAACGCTGAATTTATATAGTCGCAAACGACGAAACTTACGCTCTGGCAGCCTAACGGCCGGCCAGACACTACAACGGTTCGCAGATGGGCCGGGGGCGTCAAAACCCTGTAGTGTCACTCTACATCTGCTAGTGCTGTTCCGGGTTACTTGGTTCAGTGCGAAATAATAGGTAACTCGCCAAAGTCCAGCCTGTCCGTCGGCGTGGCAGAGGTTAAATCCAAATGACACGACTAAGTATGTAGAACTCACTGTAGAGGACTTTCGGACGCGGGTTCAACTCCCGCCAGCTCCACCA

TABLE 39 tmDNA Sequence for Hydrogenophaga palleroni (beta-purple)GGGGCTGATTCTGGATTCGACGTGGGTTCGGACGCGCAGCAGGGCATGTCGAGGTTCTGTCACC (SEQ IDNO: 47) TCGTAAATCAGCAGAAAAAAACCAACTGCAAACGACGAACGTTTCGCACTCGCCGCTTAAACACCGGTGAGCCTTGCAACAGCAGGCCGATGGGCTGGGCAAGGGGGTCGCAAGACCTCCCGGCTGCAAGGTAATTTACATCGGCTGGTTCTGCGTCGGGCACCTTGGCGCAGGATGAGATTCAAGGATGCTGGCTTCCCGTTTAGCGTGCCACTGCGCGACTCGGGCGGCGAGACCCAAATCAGACGGCTACACATGTAGAACTGCTCGAAAAAGGCTTGCGGACGGGGGTTCAACTCCCGCCAGCTCCACCA

TABLE 40 tmDNA Sequence for Methylobacillus glycogenes (beta-purple)GGGGGCGGAAAGGATTCGACGGGGGTTGCAAAGCAGCGCAGGGCATACCGAGGCCTAGTCACCT (SEQ IDNO: 48) CGTAAATAAACTAGAACAAGTATAGTCGCAAACGACGAAACTTACGCTCTAGCCGCTTAATCCCGGCTGGACGCTGCACCGAAGGGCCTCTCGGTCGGGTGGGGTAACCCACAGCAGCGTCATTAAGAGAGGATCGTGCGATATTGGGTTACTTAATATCGTATTAAATCCAAGGTAACTCGCCTGCTGTTTGCTTGCTCGTTGGTGAGCATCAGGTTAAATCAAACAACACAGCTAAGTATGTAGAACTGTCTGTGGAGGGCTTGCGGACGGGGGTTCGATTCCCGCCGCCTCACCACCA

TABLE 41 tmDNA Sequence for Nitrosomonas cryotolerans (beta-purple)GGGGCTGATTCTGGATTCGACGTGGGTTGCAAAGCAGCGCAGGGCATACCGAGGACCAGAATAC (SEQ IDNO: 49) CTCGTAAATACATCTGGAAAAAAATAGTCGCAAACGACGAAAACTACGCTTTAGCCGCTTAATACGGCTAGCCTCTGCACCGATGGGCCTTAACGTCGGGTCTGGCAACAGACAGCAGAGTCATTAGCAAGGATCGCGTTCTGTAGGGTCACTTTACAGAACGTTAAACAATAGGTGACTCGCCTGCCATCAGCCCGCCAGCTGGCGGTTGTCAGGTTAAATTAAAGAGCATGGCTAAGTATGTAGAACTGTCTGTAGAGGACTTGCGGACGCGGGTTCAACTCCCGCCAGTCCACCA

TABLE 42 tmDNA Sequence for Pseudomonas testosteroniGGGGCTGATTCTGGATTCGACGTGGGTTCGGGACCGGTGCGGTGCATGTCGAGCTTGAGTGACG (SEQ IDNO: 50) CTCGTAAATCTCCATTCAAAAAACTAACTGCAAACGACGAACGTTTCGCACTCGCCGCTTAATCCGGTGAGCCTTGCAACAGCACGCTAGTGGGCTGGGCAAGGGGGTAGCAATACCTCCCGGCTGCAAGGGAATTTTCATTAGCTGGCTGGATACCGGGCTTCTTGGTATTTGGCGAGATTTTAGGAAGCTGGCTACCCAAGCAGCGTGTGCCTGCGGGGTTTGGGTGGCGAGATTTAAAACAGAGCACTAAACATGTAGATCTGTCCGGCGAAGGCTTACGGACGCGGGTTCAACTCCCGCCAGCTCCACCA

TABLE 43 tmDNA Sequence for Ralstonia pickettii (Burkholderia)GGGGGCGGAAAGGATTCGACGGGGGTTGCGAAGCAGCGGAGGGCATACCGAGGACCCGTCACCT (SEQ IDNO: 51) CGTTAATCAATGGGAATGCAATAACTGCTAACGACGAACGTTACGCACTGGCAGCCTAAGGGCCGCCGTCCTCGCACTGGCTCGCTGACGGGCTAGGGTCGCAAGACCAGCGAGGTCATTTACGTCAGATAAGCTTTAGGTGAGTCACGGGCCTAGAGACGAAAACTTAGTGAATCGCCGTCGTAGAGCGTGTTCGTCCGCGATGCGGCGGTTAAATCAAATGACAGAACTAAGTATGTAGAACTCTCTGTGGAGGGCTTGCGGACGCGGGTTCGATTCCCGCCGCCTCACCACCA

TABLE 44 tmDNA Sequence for Variovaxparadoxus (pseudomonas sp.) (SEQ IDNO:52) GGGGCTGATTCTGGATTCGACGTGGGTTCGGAGTCGCAGCGGGGCATGTCGAGCTGAATGCGCTCGTAAAACAGATTCAAACAAACTAACTGCAAACGACGAACGTTTCGCACTCGCTGCTTAATTGCCAGTGAGCCTTGCAACAGTTGGCCGATGGGCTGGGCAAGGGGGTCTGGAGCAATCCTGACCTCCCGGCTGCAAGGATAACTACATGGGCTGGCTCCGATCCGGGTACCTTGGGTCGGGGCGAGAAAATAGGGTACTGGCGTCCGGTTTAGCGTGTGACTGCGCGACTCCGGAAGCGAGACTCAAAACAGATCACTAAACATGTAGAACTGCGCGATGAAGGCTTGCGGACGGGGGTTCAACTCCCGCCAGCTCCACCA

TABLE 45 tmDNA Sequence for Bdellovibrio bacteriovorus (deltaproteobacterie) (SEQ ID NO:53)GGGGGCGGAAAGGATTCGACGGGGGTGCTGAAGCATAAGGAGCATACCGGGGCGGATGAGGACCTCGTTAAAAACGTCCACTTTGTAATTGGCAACGATTACGCACTTGCAGCTTAATTAAGCAGCACGATCAACCTTGTGGTGGTTCCGCACTTGGATTGATCGTCATTTAGGGACCTCGGCGTGTTGGGTTTTCTCCAGCAGACATGCTTAAATTTACTGGGGGAGAGGTCTTAGGGATTTTGTCTGTGGAAGCCCGAGGACCAATCTAAAACACTGACTAAGTATGTAGCGCCTTATCGTGGATCATTTGCGGACGGGGGTTCGATTCCCGCCGCCTCCACCA

TABLE 46 tmDNA Sequence for Myxococcus xanthus (delta proteobacterie)(SEQ ID NO: 54) GGGGGCGGAAAGGATTCGACGGGGGCATTGAAGTTCGAGACGCGTGCCGAGCTTGTCAGGTAGCTCGTAAATTCAACCCGGCAAAGACACAAAAGCCAACGACAACGTTGAGCTCGCGCTGGCTGCCTAAAAACAGCCCATAGTGCGCGGTCCCCCCGCCCTCGGCCTGTGGGGTTGGGACAGACCGTCATAATGCAGGCTGGCTGCCGAGGGTGCCTGGACCCGAGGTGGCGAGATCTTCCCAGGACCGGCTCTGAGTATCCCGTCCGTGGGAGCCTCAGGGACGTAGCAAATCGCGGACTACGCACGTAGGGTCGAAGAGCGGACGGCTTTCGGACGCGGGTTCGATT CCCGCCGCCTCCACCA

TABLE 47 tmDNA Sequence for Sulfurospirillum Deleyianum (SEQ ID NO:55)GGGGCTGATTCTGGATTCGACAGGAGTAGTTTTAGCTTATGGCTGCATGTCGGGAGTGAGGGTCTTCCGTTACACAACCTTCAAACAATAACTGCTAACAACAGTAACTATCGTCCTGCTTACGCGCTAGCTGCGTAAGTTTAACAAATAATGGACTGCTCTCCCCTTTGATGCTATCTTAGGAGGTCTTGGAGAGTATCATAGATTTGATAGCTATATTACATGAACGCCTTTACATGTAATGAAGTTAAAGGCTCGTTTTGCGTAGTTTTCTGATTGTTGTACGAAGCAAAATTAAACACTATCAACAATATCTAAGCATGTAGACGTCATAGGTGGCTATTTTTGGACTGCGGGTTCAACTCCCGCCAGCTCCACCA

TABLE 48 tmDNA Sequence for Chromatium vinosum (SEQ ID NO: 56)GGGGCTGATTCTGGATTCGACGTGGGTCGCGAAACCTAAGGTGCATGCCGAGGTGCGGTTGACCTCGTAAAACCCTCCGCAAACTTATAGTTGCCAACGACGACAACTACGCTCTCGCTGCTTAATCCCAGCGGGCCTCTGACCGTCACTTGCCTGTGGGCGGCGGATTCCAGGGGTAACCTCACACAGGATCGTGGTGACGGGAGTCCGGACCTGATCCACTAAAACCTAACGGAATCGCCGACTGATCGCCCTGCCCTTCGGGCGGCAGAAGGCTAAAAACAATAGAGTGGGCTAAGCATGTAGGACCGAGGGCAGAGGGCTTGCGGACGCGGGTTCAACTCCCGCCA GCTCCACCA

TABLE 49 tmDNA Sequence for Pseudomonas fluorescens (gammaproteobacteria) (SEQ ID NO:57)GGGGCTGATTCTGGATTCGACGCCGGTTGCGAACCTTTAGGTGCATGCCGAGTTGGTAACAGAACTCGTAAATCCACTGTTGCAACTTTCTATAGTTGCCAATGACGAAACCTACGGGGAATACGCTCTCGCTGCGTAAGCAGCCTTAGCCCTTCCCTCCTGGTACCTTCGGGTCCAGCAATCATCAGGGGATGTCTGTAAACCCAAAGTGATTGTCATATAGAACAGAATCGCCGTGCAGTACGTTGTGGACGAAGCGGCTAAAACTTACACAACTCGCCCAAAGCACCCTGCCCGTCGGGTCGCTGAGGGTTAACTTAATAGACACGGCTACGCATGTAGTACCGACAGCAGAGTACTGGCGGACGCGGGTTCAACTCCCGCCAGCTCCACCA

TABLE 50 tmDNA Sequence for Borrelia afzeli (SEQ ID NO:58)GGGGCTGATTCTGGATTCGACTGAAAATGCTAATATTGTAAGTTGCAAGCAGAGGGAATCTCTTAAAACTTCTAAAATAAATGCAAAAAATAATAACTTTACAAGTTCAAACCTTGTAATGGCTGCTTAAGTTAGCAGAGAGTTTTGTTGAATTTGGCTTTGAGATTCACTTATACTCTTTTAGACATCGAAGCTTGCTTAAAAATGTTTTCAAGTTGATTTTTAGGGACTTTTATACTTGAGAGCAATTTGGCGGTTTGCTAGTATTTCCAAACCATATTGCTTAGTAAAATACTAGATAAGCTTGTAGAAGCTTATAGTATTGTTTTTAGGACGCGGGTTC AACTCCCGCCAGTCCACCA

TABLE 51 tmDNA Sequence for Borrelia crociduarae (SEQ ID NO:59)GGGGCTGATTCTGGATTCGACTAAGAACTTTAGTAGCATAAATGGCAAGCAGAGTGAATCTCTTAAAACTTCTTTAATAAATGCAAAAAATAATAACTTTACAAGTTCAGATCTTGTAATGGCTGCTTAATTTAGCAGAGAGTTTTGTTGGATTTTGCTTTGAGGTTCAACTTATACTCTTTAAGACATCAAAGTATGCCTAAAAATGTTTCAAGTTGATTTTTAGGGACCTTTAAACTTGAGAGTAATTTGGTGGTTTGCTTGTTTTCCAAGCCTTATTGCTTTTTCTAAAAATTAGCTAAGCTTGTAGATATTTATGATATTATTTTTAGGACGCGGGTTCAACTCCC GCCAGTTCCACCA

TABLE 52 tmDNA Sequence for Borrelia hermsii (SEQ ID NO:60)GGGGCTGATTCTGGATTCGACTAAAAACTTTAGTAGCATAAATTGCAAGCAGAGGGAATCTCTTAAAACTTCTTTAATAAATGCAAGAAATAATAACTTTACAAGTTCAAATCTTGTAATGGCTGCTTAAATTAGCAGAGAGTTCTGCTGGATTTTGCTTTGAGGTTCAGCTTATACTCTTTTAAGACATCAAAGCTTGCTTAAAAATATTTCAAGTTGATTTTTAGGGACTTTTAAATTTGAGAGTAATTTGGCGGTTTGCTAGTTTTTCCAAACCTTATTACTTAAAGAAAACACTAGCTAAGCTTGTAGATATTTATGATATTATTTTTAGGACGCGGGTTCAACTC CCGCCAGCTCCACCA

TABLE 53 tmDNA Sequence for Borrelia garinii (SEQ ID NO: 61)GGGGCTGATTCTGGATTCGACTGAAAATGCGAATATTGTAAGTTGCAGGCAGAGGGAATCTCTTAAAACTTCTAAAATAAATGCAAAAAATAATAACTTTACAAGCTCAAACCTTGTAATGGCTGCTTAAGTTAGCAGGGAGTTTCGTTGAATTTGGCTTTGAGGTTCACTTATACTCTTTTCGATATCGAAGCTTGCTTAAAAATGTTTTCAAGTTAATTTTTAGGGACTTTTGTACTTGAGAGCAATTTGGCGGTTTGCTAGTATTTCCAAACCATATTGCTTAAGTAAAATGCTAGATAAGCTTGTAGAAGCTTATAATATTGTTTTTAGGACGCGGGTTCAACTCC CGCCAGTCCACCA

TABLE 54 tmDNA Sequence for Thermodesulfobacterium commune (70 degrees)(SEQ ID NO:62) GGGGGCGGAAAGGATTCGACGGGGATAGGTAGGATTAAACAGCAGGCCGTGGTCGCACCCAACCACGTTAAATAGGGTGCAAAAACACAACTGCCAACGAATACGCCTACGCTTTGGCAGCCTAAGCGTGCTGCCACGCACCTTTAGACCTTGCCTGTGGGTCTAAAGGTGTGTGACCTAACAGGCTTTGGGAGGCTTAATCGGTGGGGTTAAGCCTCCCGAGATTACATCCCACCTGGTAGGGTTGCTTGGTGCCTGTGACAAGCACCCTACGAGATTTTCCCACAGGCTAAGCCTGTAGCGGTTTAATCTGAACTATCTCCGGACGCGGGTTCGATTCCCGCCGCCTC CCCACCA

TABLE 55 tmDNA Sequence for Thermotoga neapolitana (Thermotogales) (SEQID NO:63) GGGGGCGGAAAGGATTCGACGGGGATGGAGTCCCCTGGGAAGCGAGCCGAGGTCCCCACCTCCTCGTAAAAAAGGTGGGAACACGAATAAGTGCCAACGAACCTGTTGCTGTTGCCGCCTAATAGATAGGCGGCCGTCCTCTCCGGAGTTGGCTGGGCTCCGGAAGAGGGCGTGAGGGATCCAGCCTACCGATCTGGGCTCCGCCTTCCGGCCCGGATCGGGAAGGTTCAGGAAGGCTGTGGGAAGCGACACCCTGCCCGTGGGGGGTCCTTCCCGAGACACGAAACACGGGCTGCGCTCGGAGAAGCCCAGGGGCCTCCATCTTCNGACGCGGGTTCGATTCCCGCCAC CTCCACCA

TABLE 56 tmDNA Sequence for Deinococcus proteolyticus (SEQ ID NO:64)GGGGGCGGAAAGGATTCGACGGGGGAACGGAAAGCGCTGCTGCGTGCCGAGGAGCCGTTGGCCTCGTAAACAAACGGCAAAGCCATTAACTGGCGAAAATAACTACGCTCTCGCTGCTTAAGTGAGACAGTGACCACGTAGCCCCGCCTTTGGCGACGTGTGAACTGAGACAAAAGAAGGCTAGCTTAGGTGAGGTTCCATAGCCAAAAGTGAAACCAAATGGAAATAAGGCGGACGGCAGCCTGTTTGCTGGCAGCCCAGGCCCGACAATTTAAGAGCAGACTACGCACGTAGATGCACGCTGGATGGACCTTTGGACGCGGGTTCGATTCCCGCCAGCTCCACCA

TABLE 57 mDNA Sequence for Prosthecobacter fusiformis (verrucomicrobia)(SEQ ID NO:65) GGGGCTGATTCTGGATTCGACGGGGAGTACAAGGATCAAAAGCTGCAAGCCGAGGTGCCGTTACCTCGTAAAACAACGGCAAAAAAGAAGTGCCAACACAAATTTAGCATTAGCTGCTTAATTTAGCAGCTACGCTCTTCTAACCCGGGCTGGCAGGGTTAGAAGGGTGTCATAATGAGCCAGCTGCCCCTTCCGACTCCCCTAAGGAAGGGAAAGATGTAGGGGATAGGTGCTTACAGAATCCTGCGGGAGGGAGTCTGTAAGTGCCGAAAAGTTAAAACTCCCGCTAAGCTTGTAGAGGCTTTTGATTCTTGCTCTCTGGACGCGGGTTCAACTCCCGCCAGCTCCAC CA

TABLE 58 tmDNA Sequence for Verrucomicrobium spinosum (verrucomicrobium)(SEQ ID NO:66) GGGNNNNATTTGGAATTCGCCGAATGCTAGAAGTGGAGGCTGCATGCCGCGGATGATTCGTTGGCCGCTTTACCAATTCGGATCAAACAACTAAATGCGGACTCTAACGAGCTTGCCCTCGCCGCTTAATTGACGGTGACGTTCCTCCAGTGAAGTCTGTGAATTGGAGGAGCGACTACTTACAGGCTGGCCAAAAGAGCGGGCGACCGGCCCCAAGGCGAGATCTACAGGCCGCTGGATGGACGGCATCCTGGCAGTAGGAGGCTGGACATCGAGATCAAATNATTGCCTGAGCATGGAGACGCTTTCATAAAGGNGTTCGGACAGGG

Example 4 Alignment of tmRNA Sequences

The newly discovered tmRNA sequences and several known tmRNA sequenceswere aligned to identify target sites for drug development. Thealignments of the sequences are shown in FIGS. 3A-11B. The nucleotidesin the tmRNA sequences of these figures exist in several motifs (Feldenet al., 1999). These motifs include nucleotides considered to be in RNAhelices (Watson-Crick base-pairs GC or AU, or GU Wobble base-pairs).Nucleotides that are in single stranded RNA domains, hence notbase-paired. Some nucleotides in the single stranded domains areuniversally conserved nucleotides. Other nucleotides are the exceptionsto a quasi-sequence conservation in the sequences alignment. Severalnucleotides exist in well established non-canonical structural motifs inRNA structures; for example AG-GA pairs, AA pairs, etc. Some nucleotidesare universally conserved Wobble GU base-pairs.

All the gene sequences have been decomposed in several structuraldomains that have been indicated with names at the top of each block ofsequences. These domains are respectively from the 5′-end to the 3′-endof the sequences: H1, H5, H2, PK1, H4, PK2, PK3, PK4, H5 and H6. Thebars delineate all the structural domains. H means helices and PK meanspseudoknot. A pseudoknot is made of the pairing of parts of an RNA-loopwith an upstream sequence. Consequently, two helices are made (shown inFelden et al., 1999) for all the 4 pseudoknots PK1 to PK4 for eachsequence. Moreover, the tRNA-like domain as well as the coding sequence,namely the two functional units of the molecule, have also beenindicated for each sequence.

The sequences, especially as identified by the sequence alignment,represent targets for the development of drugs which may be broadlyapplicable to many kinds of bacteria, or may be broadly applicable onlyto a particular genera of phylum of bacteria, or may be specificallyapplicable to a single species of bacteria.

Common Structural Features for Drug Targeting:

For all the novel tmRNA sequences, as well as with the ones that arealready known, there are systematically several structural domains thatare always found. These domains can be used as targets for thedevelopment of drugs which may be genera specific or which may beeubacteria specific. These domains are either RNA helices which can besometimes interrupted by bulges or pseudoknots. The RNA helices whichare always present are H1, H2, H5 and H6. Helices H1 and H6 are found inall canonical transfer RNAs. Thus, H1 and H6 are not good targets fordrug development because drugs that would target them will alsointerfere with the biology of the individual that has a given disease.Consequently, very good candidates for development of drugs fortargeting as many bacteria as possible are helices H2 and H5. Moreover,helices H2 and H5 are critical for the folding of all these tmRNA sinceboth of them connect the two ends of the molecule together. Disruptionof either H2 or H5 with a specific drug would lead to inactive tmRNAmolecules in vivo. Similarly, pseudoknots PK1, PK2 and PK3 are alwaysfound in the bacterial tmRNAs. The PK1 structural domain is strictlyconserved in the tmRNAs and is located upstream of the coding sequence.Since these pseudoknots are not found in all canonial transfer RNAs,they can also be targeted with specific drugs. Disruption of either PK1or PK2 or PK3 with a specific drug would lead to inactive tmRNAmolecules in vivo.

Specific Structural Features in Each Phylum that could be Targeted byDrugs:

In addition to developing drugs which broadly target many bacteria,drugs are developed which are more genera specific. For trying to targetspecifically a given bacteria or a complete phylum, the coding sequence(shown in all the alignments) is a very good candidate. Indeed, thisregion of the RNA is very accessible for DNA antisense binding, whichhas been shown for Escherichia coli, and thus, is also available forinteraction with other drugs. Moreover, this is a critical functionaldomain of the molecule in its quality-control mechanism in cells. Inaddition, this coding sequence would be the ideal target to use fordesigning specific PCR-based diagnostic assays for infection diseases.

Interestingly, some structural domains are present only in a givenbacterial phyla and could be targeted for discovering a drug that willbe specific of a phylum, but not of the others. For example:

(1) in the cyanobacteria, the fourth pseudoknot PK4 is made of twosmaller pseudoknots called PK4a and PK4b;

(2) in the mycoplasma, helix H2 is made of only 4 base-pairs instead of5 in the other species;

(3) for two sequences of chlorobium as well as Bacteroidesthetaiotaomicron and ppm gingiv., there is an additional domain justdownstream of the coding sequence that is unique to them;

(4) there is always a stem-loop in the coding sequence of theactinobacteria (Felden et al., 1999); and

(5) all the beta proteobacteria possess a sequence insertion inpseudoknot PK2 (shown in the alignment).

The novel sequences described herein, when aligned, show that specificstructural domains within tmRNA are strictly conserved, as for examplepseudoknot PK1 is located upstream (at the 5′-side) of the codingsequence. As previously disclosed, this pseudoknot is a target forfuture antibacterial drugs. Moreover, recent data have shown that thisPK1 pseudoknot, among all the four pseudoknots within tmRNA genesequences (sometimes there's only 2 or 3 detectable pseudoknots,depending upon the sequences), is the only one that its correct foldingis essential for the biological activity of tmRNA (Nameki et al., 1999;Nameki et al., 2000).

While the invention has been disclosed in this patent application byreference to the details of preferred embodiments of the invention, itis to be understood that the disclosure is intended in an illustrativerather than in a limiting sense, as it is contemplated thatmodifications will readily occur to those skilled in the art, within thespirit of the invention and the scope of the appended claims.

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1. An isolated nucleic acid sequence selected from the group consistingof the tmRNA sequence for Legionella pneumophilia set forth in SEQ IDNO:155, a tmDNA sequence encoding said tmRNA sequence, and a complementof said tmDNA sequence.
 2. A method for diagnosing a bacterialinfectious agent comprising determining the presence of a bacterialnucleic acid sequence selected from the group consisting of the tmRNAsequence for Legionella pneumophilia set forth in SEQ ID NO:155, a tmDNAsequence encoding said tmRNA sequence, and a complement of said tmDNAsequence.
 3. The method of claim 2, wherein the determination is made byperforming an amplification-based assay.
 4. The isolated nucleic acidsequence of claim 1, wherein the nucleic acid sequence is the tmDNAsequence encoding the tmRNA sequence for Legionella pneumophilia setforth in SEQ ID NO:155.
 5. The method of claim 2, wherein the bacterialnucleic acid sequence is the tmDNA sequence encoding the tmRNA sequencefor Legionella pneumophilia set forth in SEQ ID NO:155.
 6. The method ofclaim 3, wherein the bacterial nucleic acid sequence is the tmDNAsequence encoding the tmRNA sequence for Legionella pneumophilia setforth in SEQ ID NO:155.